Despite substantial degradation, 2-arachidonoylglycerol is a potent full efficacy agonist mediating CB(1) receptor-dependent G-protein activation in rat cerebellar membranes

Abstract

1. Two endocannabinoids, arachidonoyl ethanolamide (AEA) and 2-arachidonoylglycerol (2-AG) bind and activate G-protein-coupled cannabinoid receptors, but limited data exist on their relative ability to activate G-proteins. 2. Here we assess agonist potency and efficacy of various cannabinoids, including 2-AG, HU-310 (2-arachidonoyl glyceryl ether, a third putative endocannabinoid), HU-313 (another ether analogue of 2-AG), AEA, R-methanandamide (an enzymatically stable analogue of AEA), and CP-55,940 at rat brain CB(1) receptors using agonist-stimulated [(35)S]-GTPgammaS binding to cerebellar membranes and whole brain sections. Degradation of endocannabinoids under experimental conditions was monitored by HPLC. 3. To enhance efficacy differences, agonist dose-response curves were generated using increasing GDP concentrations. At 10(-6) M GDP, all compounds, except HU-313, produced full agonists responses approximately 2.5 fold over basal. The superior efficacy of 2-AG over all other compounds became evident by increasing GDP (10(-5) and 10(-4) M). 4. In membrane incubations, 2-AG was degraded by 85% whereas AEA and HU-310 were stable. Pretreatment of membranes with phenylmethylsulphonyl fluoride inhibited 2-AG degradation, resulting in 2 fold increase in agonist potency. Such pretreatment had no effect on AEA potency. 5. Responses in brain sections were otherwise consistent with membrane binding data, but 2-AG evoked only a weak signal in brain sections, apparently due to more extensive degradation. 6. These data establish that even under conditions of substantial degradation, 2-AG is a full efficacy agonist, clearly more potent than AEA, in mediating CB(1) receptor-dependent G-protein activity in native membranes.

Figure 1

Dose-response curves and maximal efficacy (E_max) of cannabinoid ligands in activating G-proteins in rat cerebellar membranes at various GDP concentrations. The data represent the mean±s.e.mean of [³⁵S]-GTPγS binding over basal from three to four independent experiments performed in duplicate. When not visible, the error bars fell within the size of the symbol.

Figure 2

2-AG activates G-proteins in rat cerebellar membranes via CB₁ receptors. Membranes were incubated in the presence of 10⁻⁶ M 2-AG together with increasing concentrations of the CB₁ receptor antagonist SR141716 or with 10⁻⁶ M SR144528 (a CB₂ receptor antagonist) as indicated. The data represent the mean of [³⁵S]-GTPγS binding over basal±s.e.mean from at least three independent experiments performed in duplicate.

Figure 3

[³⁵S]-GTPγS autoradiography reveals CB₁ receptor-dependent G-protein activity in rat brain sections. Adjacent sagittal sections, obtained from 4-week-old male Wistar rats, were processed for [³⁵S]-GTPγS autoradiography, as described in Methods using a 3-step incubation protocol. This consisted of preincubation (step 1), loading with 2 mM GDP (step 2) and [³⁵S]-GTPγS binding for 45 min at 20°C (step 3). Cannabinoids (5×10⁻⁵ M) were present during step 3, whereas the A₁ receptor antagonist DPCPX (10⁻⁶ M) was present throughout steps 2 and 3. Note weak responses to 2-AG (most evident in the substantia nigra) and the completely overlapping distribution of activated G-proteins in response to all five cannabinoids, especially in the cerebellar molecular cell layer (Cbm), cerebral cortex (Cx), hippocampus (Hi), substantia nigra (SN) and striatum (Str). Scale bar=2 mm.

Figure 4

Degradation of 2-AG during incubations mimicking [³⁵S]-GTPγS binding assays in cerebellar membranes and brain sections. The data represent the mean (±s.d. n=3) of relative (%) peak areas for each compound.

Figure 5

Pretreatment of cerebellar membranes with PMSF inhibits 2-AG degradation with concomitant increase in G-protein activation potency. Membranes were incubated for 30 min at 25°C in the presence or absence of 10⁻³ M PMSF, followed by 90 min incubation with 2-AG (5×10⁻⁵ M) to study degradation (A) or with the indicated concentrations of 2-AG, 1-AG or AEA to assess CB₁ receptor-dependent G-protein activity (B). Note significant inhibition of arachidonic acid (AA) formation and concomitant accumulation of 2-AG and 1-AG in PMSF-treated membranes. Note also significant leftward shifts in dose-response curves for 2-AG and 1-AG and total lack of PMSF in the case of AEA. For (A) the data represent the mean (±s.d. n=3) of relative (%) peak areas for each compound. For (B) the data represent the mean±s.e.mean of [³⁵S]-GTPγS binding over basal from two (AEA and 1-AG) to four independent experiments performed in duplicate. When not visible, the error bars fell within the size of the symbol. An asterisk denotes significant difference between pairs (P<0.05).